The primary objective of this investigation is to determine how various protein factors act to regulate gene expression in Escherichia coli and bacteriophage-infected E. coli at the level of RNA transcription. The major emphasis will be on the structure, function and mechanism of action of the rho transcription termination protein and in particular on the function of its ATPase activity. The specific aims of the project are: to study interactions between rho protein and RNA, RNA-DNA complexes, RNA polymerase, transcription complexes and ATP; to demonstrate that rho is responsible for polar effects in gene expression; and to demonstrate that rho acts as part of a feedback loop that couples the level of transcription of an operon with the level of translation of the mRNA synthesized. The majority of the analyses will be made with highly purified components and with in vitro assays. Standard biochemical methodology will include enzyme assays, binding measurements, affinity labeling, protease and nuclease susceptibility, RNA and DNA sequence analysis, hybridization of radiolabeled RNA to DNA restriction nuclease fragments, and polyacrylamide gel electrophoresis analysis of protein synthesized in vitro. The insights gained from understanding the fundamental mechanism of gene regulation will help in understanding the etiology of diseases that result from alteration of normal gene regulation such as with cancers and virus infections.